Method for treating cystic fibrosis

ABSTRACT

The present invention provides a method of identifying CFTR-binding compounds for treating cells having a reduced apical Cl −  conductance, such as cystic fibrosis cells. This identification method involves the use of polypeptide Iα, which constitutes a portion of the CFTR protein. The present invention also provides a method of treating CF cells by contacting cells having a reduced apical Cl −  conductance with a therapeutically effective quantity of a compound selected by the present inventive identification method. Preferred compounds for such treatment have little or no affinity for adenosine cell receptors. The present invention provides novel compounds useful in practicing the present inventive method, as well as pharmaceutical compositions containing such compounds.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation-in-part of copending application Ser. No.07/952,965, filed Sep. 29, 1992, now U.S. Pat. No. 5,366,977.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of identifyingCFTR-binding compounds for treating cells having a reduced apical Cl⁻conductance, such as cystic fibrosis cells. The present inventionfurther relates to compounds and pharmaceutical compositions thereofuseful in treating such cells.

BACKGROUND OF THE INVENTION

[0003] Healthy animal cells require, among other conditions andmaterials, the movement of various inorganic ions across the cellmembrane to be maintained such that a proper balance of the ions providethe requisite electrical potential across the cellular membrane as wellas a life-promoting internal ionic strength. For example, Na⁺, Cl⁻, K⁺,and Ca⁺⁺ are known to cross cell membranes in animals such that K⁺ andCa⁺⁺ are accumulated intracellularly to a varying extent in differentcells at different times of development, whereas Na⁺, in large measure,is excluded from the interior of a cell. The cross-cellular movement ofthese ions is mediated by Na⁺/K⁺- and Ca⁺⁺-dependent ATPases that aremembrane bound at the sites of appropriate ion channels. Chloride ionwas believed to permeate animal cells by passive means to equilibrate inconcentration between the external and internal fluid, resulting in anunderrepresentation of Cl⁻ intracellularly in consequence of the overallnegative intracellular change. Conductance of chloride, however, hasbeen shown to be mediated actively as well, by means of a Cl⁻ channel(see Edwards, Neuroscience, 7, 1335-1366 (1982)).

[0004] Results from research directed to the pathology of cysticfibrosis (“CF”) has provided information on the ill-effects that an ionconductance impairment at the cellular level can have on a person'shealth, at many levels. CF was known to have a genetic basis because ofits differential incidence among white Americans (between 1/1600 and1/2000 live births) as compared to African Americans (about 1/17,000live births). Indeed, research over the past decade has revealed that aheritable discrete gene mutation is associated with the clinicalsymptoms of CF, including abnormal exocrine gland and lung functions.

[0005] More specifically, CF is caused by mutations in the cysticfibrosis transmembrane regulator (CFTR) gene, the most common of whichis the deletion of a phenylalanine residue at position 508 (Phe⁵⁰⁸; seeFIG. 1 for a physical map of the CFTR protein). The mutated CFTR proteinis referred to as ΔF508, the site of which is within the firstnucleotide binding fold (NBF-1). Schoumacher et al., Proc. Natl. Acad.Sci., 87, 4012-4016 (1990); Riordan et al., Science, 245, 1066-1073(1979). More specifically, this mutation is located in a lengthyinternal segment of the NBF-1, flanked on the N-terminal side by theWalker A sequence (amino acid position 458-471; also referred to as“CFTR[458-471]”) and on the C-terminal side by the contiguous C domain(amino acid position 548-560; also referred to as “CFTR[548-560]”) andthe Walker B domain (amino acid position 561-573; also referred to as“CFTR[561-573]”). The physiological role of the intervening sequencelocated between CFTR[471] and CFTR[561], which includes the polypeptidesequences of Iα, Iβ, and Io (see FIG. 1), is unknown.

[0006] Certain mutations in the CFTR gene, such as that resulting inΔF508, cause an abnormal potential difference across CF epithelia. Theabnormality is due to a reduced cellular apical chloride (Cl⁻)conductance. Consequently, chloride and sodium transport acrossepithelial membranes of an individual afflicted with CF, for example, isabnormal. It is also known that cells carrying the ΔF508 mutation havehigher than normal CFTR protein bound to their endoplasmic reticulum(hereinafter, “ER”), although wild type cells also retain and degrade asubstantial amount of CFTR protein in their ER, albeit much less so thanthe ΔF508 mutant. Ward et al., J. Gen. Physiol., 104, 33a (1994).

[0007] This CFTR mutation apparently is responsible forpathophysiological changes in the respiratory system, among others.Nearly all patients suffering from the disease develop chronicprogressive disease of the respiratory system. In the majority of cases,pancreatic dysfunction occurs, and hepatobiliary and genitourinarydiseases are also frequent.

[0008] Although survival of cystic fibrosis patients has improved inrecent years, the median survival is still only about 28 years despitethe development and implementation of intensive supportive andprophylactic treatment. Present efforts to combat the disease havefocused on drugs that are capable of either activating the mutant CFTRgene product or otherwise causing additional secretion of Cl⁻ fromaffected cells. Gene therapy is another area of active research, whereinthe anion conductance deficit may be repaired by the introduction of arecombinant wild-type CFTR gene, i.e., a CFTR gene that lacks a mutationthat results in the abnormality.

[0009] Encouraging clinical results have been reported recently for theuse of aerosols containing either amiloride (Knowles et al., N. Engl. J.Med., 322, 1189-1194 (1990)) or a mixture of ATP and UTP (Knowles etal., N. Engl. J. Med., 325, 533-538 (1991)), which slow the accumulationof Cl⁻ in the epithelium of the trachea.

[0010] Other drugs that purportedly are useful in the treatment of CFhave been described. For example, U.S. Pat. No. 4,866,072 describes theuse of9-ethyl-6,9-dihydro-4,6-dioxo-10-propyl-4H-pyrano(3,2-g)quinoline-2,8-dicarboxylicacid or a pharmaceutically acceptable derivative thereof in thetreatment of CF. U.S. Pat. No. 4,548,818 describes the use of a3-alkylxanthine to treat chronic obstructive pulmonary disease (COPD).U.S. Pat. No. 5,032,593 describes the use of a 1,3-alkyl substituted8-phenylxanthine or a pharmaceutically acceptable salt thereof in thetreatment of bronchoconstriction. U.S. Pat. No. 5,096,916 describes theuse of an imidazoline α-adrenergic blocking agent and vasodilator, suchas tolazoline, in the treatment of COPD, including cystic fibrosis,chronic bronchitis and emphysema, or COPD in association with asthma.

[0011] Historically, theophylline has been administered to asthmatic andCF patients to enhance lung function. Such lung function enhancement iscaused principally by bronchodilation, which is due to the action oftheophylline on smooth muscles and inflammation. Theophylline has beenshown not only to inhibit phosphodiesterase, but also to antagonizeadenosine receptors. Accordingly, because theophylline acts at more thanone site, it obviously lacks specificity, thus reducing its usefulnessto treat CF. In view of the fact that antagonism of the A₁ adenosinereceptor, not inhibition of phosphodiesterase, has been shown to resultin stimulating chloride efflux from CF cells, such lack of specificitycould result in undesired side effects, such as detrimental effects tocardiac, renal, and/or central nervous system tissue. In addition, largedoses of theophylline must be administered to achieve a beneficialeffect, thus increasing the risk of side effects from the high toxicityof the compound.

[0012] Other compounds that resemble theophylline in basic structurehave been tested but have not been found to be useful in evokingchloride efflux from CF cells and, therefore, has no or little potentialin the treatment of cystic fibrosis. For example, 3-isobutylmethylxanthine (IBMX), which is structurally similar to theophylline, isnonspecific in activity and highly toxic and, therefore, lacks utilityin the treatment of CF. Also ineffective in the activation of chlorideefflux are the compounds 2-thio-8-cylcopentyl-1,3-dipropylxanthine(2-thio-CPX), 1,3-dipropyl-8-noradamantylxanthine (KW-3902), and1,3-dimethyl-8-cyclopentylxanthine (CPT) (see U.S. Pat. No. 5,366,977).Similarly, substitution of the propyl group at position 1 or 3 of CPX(1,3-dipropyl-8-cyclopentylxanthine) with a one-carbon group generates acompound that is ineffective in activating chloride efflux from CFcells. Clearly, minor structural differences have a significant, if notsubstantial, impact on the effectiveness of a given compound in thetreatment of CF. Accordingly, it will be particularly useful if a methodwere available for screening compounds for an ability to promotechloride ion conductance in affected cells, which, in turn, would be acandidate therapeutic agent for treatment of cystic fibrosis patients.

[0013] The '977 patent, cited above, and Eidelman et al., Proc. Natl.Acad. Sci. USA, 89, 5562-5566 (1992), disclosed that CPX is a potent A₁adenosine antagonist that promotes chloride efflux from a humanepithelial cell line (CFPAC-1). The CFPAC-1 line, further described inExample 6, expresses the aforementioned CFTR ΔF508 mutation and can beviewed as a first generation screening material for CF-active compounds.Although CPX and its related xanthine amino congeners disclosed in the'977 patent have been shown to be relatively non-toxic and thereforepotentially useful for CF treatment, the fact that such compounds havean antagonistic effect on A₁-adenosine receptors indicates that suchtreatment probably will have an additional impact on an animal that isunrelated to the CF affliction. Accordingly, use of such compounds,indeed any compound known to have multiple targets of activity,preferably is avoided because the use of such compounds is, at least,potentially detrimental.

[0014] A drug of high potency, low toxicity, and little or nospecificity for adenosine receptors, thus, would be a highly desirableand promising therapeutic agent for the treatment of cells having areduced apical Cl⁻ conductance, such as cystic fibrosis cells. Such adrug would not only find utility in the treatment of cystic fibrosis perse but would be therapeutically useful in the treatment of COPD, ingeneral.

[0015] It is an object of the present invention to provide a method foridentifying compounds that can activate an impaired chloride conductancechannel. It is also an object of the present invention to provide amethod of correcting the reduced Cl⁻ conductance of cells that areimpaired in such conductance. It is another object of the presentinvention to provide a method of treating cystic fibrosis cells. It isyet another object of the present invention to provide a method oftreating cystic fibrosis cells having a deletion involving phenylalanineat amino acid position 508 of the cystic fibrosis transmembraneregulator. It is also an object of the present invention to providecompounds useful in such methods, in particular novel xanthinederivatives that have little or no affinity for the adenosine receptorsbut have an ability nonetheless to ameliorate the Cl⁻ imbalance of CFcells by stimulating Cl⁻ efflux.

[0016] These and other objects and advantages of the present invention,as well as additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0017] The present invention provides certain CFTR polypeptides, such asIα [SEQ ID NO:1], that exhibit an ability to bind specifically tocompounds that are known to activate the CFTR-linked chloride ion effluxin ΔF508 mutant cells and not to bind to compounds that are known not tohave such a capability to activate CFTR-linked chloride ion efflux.Moreover, the present invention provides a method for the identificationof further compounds capable of activating the CFTR-linked chloride ionefflux. Such a method includes the application of the aforementionedCFTR polypeptides as selective binding agents for ion efflux activatingcompounds. As a further refinement of the identification method, otherCFTR polypeptides, such as Iβ [SEQ ID NO:3] or Io [SEQ ID NO:4], havingamino acid sequences of adjacent or overlapping regions of the locationof Iα, are also disclosed that, despite their close or overlappingproximity to Iα, have a neutral or negative affinity for compounds knownto activate ion efflux.

[0018] Application of the aforementioned identification method may beused to identify compounds that have an ability to activate ion effluxin ion efflux deficient cells, such as CF cells, especially those havingthe ΔF508 mutation. Moreover, such compounds, in the context of thepresent invention, may be further screened to have little or no affinityfor either the A₁-adenosine cell receptor or the A₂-adenosine cellreceptor. Such compounds, accordingly, may be used in a method oftreating cells having a reduced apical Cl⁻ conductance, such as cysticfibrosis cells. Specifically, the present inventive method involvescontacting cells having a reduced apical Cl⁻ conductance with atherapeutically effective quantity of a compound that has little or noaffinity for the A₁-, A₂—, or A₃-adenosine cell receptor. Nevertheless,contact between the inventive compounds and CF cells results in Cl⁻efflux from such cells.

[0019] In particular, the method involves contacting such cells with acompound having the formula:

[0020] wherein R₁ and R₃ are the same and are C₁-C₆ alkyl or C₁-C₆alkenyl, R₇ is C₁-C₆ alkyl or hydrogen, and R₈ is C₄-C₈ cycloalkyl. Sucha compound may further be selected from the group consisting of1,3-dipropyl-7-methyl-8-cyclopentylxanthine,1,3-dipropyl-7-methyl-8-cylcohexylxanthine, cyclohexyl caffeine,1,3-diallyl-8-cyclohexylxanthine, and therapeutically effectivederivatives thereof.

[0021] The compounds used in the present inventive method resembletheophylline in basic structure; however, they differ significantly inthe substituents at the R₁, R₃, and R₈ positions. Given that minorstructural differences in compounds that resemble theophylline have beenshown to render the compounds ineffective or otherwise not useful in thetreatment of CF, it was surprising to discover that the aforementionedcompounds are effective in activating chloride efflux from CF cells.This discovery was particularly unexpected inasmuch as the apparent modeof activity of the inventive compounds does not involve any of theadenosine receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a physical map of a portion of the CFTR protein,displaying the location of the following sequences: Walker A (aminoacids 458 to 471); Iα (amino acids 477 to 508); Iβ (amino acids 509 to539); Io (amino acids 550 to 530); C (amino acids 548 to 560); andWalker B (amino acids 561 to 573).

[0023]FIG. 2 is a graph that depicts the aggregation of chromaffingranules by certain CFTR polypeptides at a concentration of 20 μg/ml(5.2 μM), at pH 6.0 and 1 mM Ca⁺⁺. The units of the x-axis representminutes of time and the units of the y-axis represent change inturbidity (Δ540 nM).

[0024]FIG. 3A is a graph that depicts the aggregation of chromaffingranules in the presence of varying concentrations of the Iαpolypeptide. The units of the x-axis represent the concentration of Iαpolypeptide (μg/ml) and the units of the y-axis represent change inturbidity (Δ540 nM). Reactions were performed as for generating the dataof FIG. 2; the initial rate was calculated for each concentration of Iα.

[0025]FIG. 3B is a Hill plot for the data displayed in FIG. 3A. TheV_(max app) was estimated from the linear extrapolation of theLineweaver-Burk plot (log (V_(i)/V_(m)−V_(i)) versus log [Iα]) of thedata, and the ratio of velocities was computed for each condition. Theslope (2.6) is the Hill coefficient (n_(H)).

[0026]FIG. 4A is a graph that depicts phosphatidylserine liposomeaggregation (y-axis is ΔA₃₅₀/min) by Iα at different pH values (x-axis).The concentration of Iα is 2 μg/ml (0.6 μM).

[0027]FIG. 4B is a graph that depicts phosphatidylserine liposomeaggregation (y-axis is ΔA₃₅₀/min) by lower concentrations of Iαpolypeptide measured over time α-axis in minutes).

[0028]FIG. 5 is a graph that depicts the effect of CPX (x-axis in nM) onaggregation of phosphatidylserine liposomes driven by Iα polypeptide(y-axis is ΔA₃₅₀/20 min). The inset graph depicts the concentrationdependence for CPX activation at the 20 minute time point, wherein they-axis is ΔA₃₅₀/20 min and the x-axis is concentration of CPX in nM.

[0029]FIG. 6 is a graph that depicts the effect of caffeine (x-axis isconcentration in nM) on aggregation of phosphatidylserine liposomesdriven by Iα polypeptide (measured on the y-axis as ΔA₃₅₀/20 min). Theinset graph depicts the concentration dependence for CPX activation atthe 20 minute time point, wherein the y-axis is ΔA₃₅₀/20 min and thex-axis is in nM concentration units of caffeine.

[0030]FIG. 7 is a graph depicting the effect of DAX concentration(x-axis is nM) on aggregation of phosphatidylserine liposomes (y-axis,ΔA₃₅₀/20 min) driven by Iα polypeptide. The inset graph depictsconcentration dependence. for DAX activation at the 20 minute timepoint, wherein the x-axis is nM units of concentration of DAX and they-axis is in units of ΔA₃₅₀/20 min.

[0031]FIG. 8 is a graph that depicts the comparison of CPX and DAXactivation of phosphatidylserine liposome aggregation by Iα polypeptide.The x-axis is in nM concentration units of CPX or DAX and the y-axis isin units of ΔA₃₅₀/20 min.

[0032]FIG. 9 depicts a graph of the results of [³⁶Cl]⁻ -efflux studiesof CFPAC cells when placed in contact with compound 1 (theophylline).The x-axis represents the nanomolar concentration of compound 1, and they-axis represents the rate of chloride efflux as a percentage ofcontrol.

[0033]FIG. 10 depicts a graph of the results of [³⁶Cl]⁻ efflux studiesof CFPAC cells when placed in contact with compound 5 (CPX). The x-axisrepresents the nanomolar concentration of compound 5, and the y-axisrepresents the rate of chloride efflux as a percentage of control.

[0034]FIG. 11 depicts a graph of the results of [³⁶Cl]⁻ efflux studiesof CFPAC cells when placed in contact with compound 22(8-cyclohexylmethyl caffeine). The x-axis represents the nanomolarconcentration of compound 22, and the y-axis represents the rate ofchloride efflux as a percentage of control.

[0035]FIG. 12 depicts a graph of the results of [³⁶Cl]⁻efflux studies ofCFPAC cells when placed in contact with compound 17(1,3-diallyl-8-cyclohexylxanthine). The x-axis represents the nanomolarconcentration of compound 17, and the y-axis represents the rate ofchloride efflux as a percentage of control.

[0036]FIG. 13 depicts a graph of the result of chloride efflux studieswhere varying the size of the 8-cycloalkyl substituent (x-axis) of atheophylline derivative is tested for its impact on K_(i) value (μM,y-axis). Data of the affinity of 8-cycloalkyltheophylline derivatives onrat cortical A₁-adenosine receptors () and rat striatedA_(2a)-adenosine receptors (Δ) are shown.

[0037]FIG. 14 depicts a graph of the result of chloride efflux studieswhere varying the size of the 8-cycloalkyl substituent (x-axis) of acaffeine derivative is tested for its impact on K_(i) value (μM,y-axis). Data of the affinity of 8-cycloalkyl caffeine derivatives onrat cortical A₁-adenosine receptors () and rat striatedA_(2a)-adenosine receptors (Δ) are shown.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention provides polypeptides that have a positiveaffinity for compounds that activate chloride ion efflux in CFTR-mutatedcells. The polypeptides of the present invention are either a portion ofthe CFTR protein, namely the segment identified in FIG. 1 as Iα, or avariant thereof having no significant difference in its functioning, asdisclosed herein. Iα is defined as the polypeptide that spans from aminoacid position 477 to amino acid position 508, and has the followingsequence: [SEQ ID NO:1] NH₂-PSEGKIKHSGRISFCSQFSWIMPGTIKENEEF-COOH

[0039] wherein the capital letters between the NH₂ and COOH groups arethe conventional single letter symbols for various amino acids, whichprotein-related nomenclature is used hereinbelow for SEQ ID NO:3 and SEQID NO:4 as well.

[0040] Polypeptide Iα [SEQ ID NO:1] may be prepared by (1) analytical orpreparative polypeptide synthesis using methods well-known in the art(see, e.g., Atherton et al., Solid Phase Polypeptide Synthesis, APractical Approach (Oxford Univ. Press, 1989)), (2) by appropriateincubation of a microorganism containing a suitable sequence of DNA orRNA for expression of polypeptide Iα using methods of geneticengineering that are well-known in the art (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, (2d ed. 1989)), or (3) by anyother suitable means. It is contemplated within the context of thepresent invention that conservative amino acid changes that, asunderstood within the-art, neither present charge nor conformationalchanges to the polypeptide, constitute further embodiments of thepresent invention. For example, it is believed that the exchange of anisoleucine for a leucine residue would have little or no effect on thefunctionality of the polypeptide Iα. It is also contemplated thatfragments of Iα may be functional as well, and thus such fragmentsdefine further embodiments of the present invention.

[0041] Suitable sequences of DNA and/or RNA for constructing a cloningapproach to synthesis of Iα may be identified by reverse translation andtranscription of the amino acid sequence of Iα [SEQ ID NO:1], using thegenetic code. Either or both of the DNA strands, i.e., the “sense” and“anti-sense” strands, may be usefully employed in the geneticengineering of clones for synthesis of Iα. Similarly, the RNA sequencemay constitute the “sense” or “anti-sense” strands, as well.Accordingly, the present invention may be viewed as embodied in the RNAor DNA sequences recited and/or referred to herein.

[0042] Any suitable means may be applied for generating the —RNAmolecules having any of the following sequences: [SEQ ID NO:2] CCN UCNGAR GGN AAR AUH AAR CAY UCN GGN CGN AUH UCN UUY UGY UCN CAR UUY UCN UGGAUH AUG CCN GGN ACN AUH AAR GAR AAY GAR GAR UUY

[0043] wherein A is adenine, G is guanine, U is uracil, C is cytosine, Wis A or U, Y is C or U, R is A or G, H is A, C or U, and N is G, A, C orU, and the symbols are organized in triplets to reflect the codons astranslated in an organism. This RNA-related nomenclature is usedhereinbelow for SEQ ID NO:5 and SEQ ID NO:6 as well.

[0044] Other RNA sequences that, upon translation, provide the samepolypeptide sequence or its functional equivalent are contemplatedhereby as well. For example, the triplet UCN can be replaced by thetriplet AGY and the triplet CGN can be replaced by the triplet AGR, andyet result in no change in the resultant polypeptide sequence. Inaddition to the neutral sequence differences identified at the RNAlevel, as provided herein, nucleotide changes that provide conservativechanges to the resultant amino acid sequence, as discussed above, arecontemplated as well. Thus, for example, the triplet AUH, which encodesthe amino acid isoleucine, may be replaced by the triplet CUN, whichencodes the isomeric amino acid leucine, without significant effect onthe functioning of the claimed polypeptide. Any of such sequences may beligated into a suitable nucleic acid vector for propagation andexpression in a suitable cellular or viral host, using methodswell-known in the art.

[0045] The genetic complement of the aforestated RNA molecules, i.e.,the anti-sense RNA, and the DNA counterparts to the sense and anti-senseRNA molecules are contemplated as embodiments of the present invention.Suitable means for generating the aforementioned sequences, theircomplements, or their DNA counterparts, as well as those disclosedhereinbelow, include in vitro polynucleotide synthesis of a manual orautomated nature. Once constructed, such a sequence may be ligated toother nucleic acid sequences that specify signals for transcription ortranslation, and to yet other nucleic acid sequences useful as a vectorfor propagation and/or expression in bacterial, viral, or animal cells.Methods for preparing such nucleic acid constructs are well-known in theart. See Sambrook et al., supra.

[0046] The polypeptide or a suitable fragment thereof may be used per seor it may be associated with another compound, such as one or more aminoacids, which may be covalently linked together as a second polypeptide,a proteoglycan, a proteolipid, a carbohydrate, or some other suitablemacromolecule. Such associations of the inventive polypeptide withanother macromolecule may be stabilized by means of covalent bonding,hydrogen bonding, adsorption, absorption, metallic bonding, van derWaals forces, ionic bonding, or other suitable inter- or intramolecularbonds or forces, or any combination thereof. For example, polypeptide Iαmay effectively be enlarged by the covalent addition of one or moreamino acids to the carboxy or amino terminus, or both termini, so longas its essential characteristic of binding to activating molecules forchloride ion efflux in CFTR-mutated cells is retained. A preferred formof the inventive polypeptide comprises the covalent linkage of thepolypeptide to a suitable affinity chromatography matrix, such ascellulose, including m-aminobenzyloxymethylcellulose,bromoacetylcellulose, carboxymethylcellulose hydrazide, and cellulosecyclic-carbonate; 6-aminohexanoic acid; sepharose, including sepharose4B, sepharose 6 MB, sepharose 6B, and thiol-sepharose 4B; beads composedof hydrophilic acrylic, polyacrylamide, and agarose; and linearpolyacrylamide.

[0047] Suitable reactive moieties, such as hydrazides, carbonates, andthe like, and suitable activators, such as cyanogen bromide,carbonyldiimidazole, chloroformates, and the like, may be applied to theaforementioned affinity chromatography matrices, using methods andmaterials well-known to the art, as provided, for example, by the 1993Sigma Chemical Company catalog at pages 1612-1618 and other sources.

[0048] Properties of the Iα polypeptide include (1) commencing orcausing the aggregation of bovine chromaffin granules in the presence ofabout 1 mM CaCl₂; having an apparent k_(1/2) of 22.2 μg/ml or 6.3 μM(see FIGS. 3A and 3B); (2) having activity in a pH range of from about5.5 to about 7.5, wherein 50% activity was measured at about 6.75 (seeFIG. 4A); (3) having a synergistic effect on commencing or causing theaggregation of liposomes when combined with a compound that activateschloride ion efflux in cells having the ΔF508 mutation, which compoundsinclude CPX, caffeine, and N,N-diallylcyclohexylxanthine (DAX) (seeFIGS. 5, 6, and 7, respectively); (4) having no increased effect oncommencing or causing the aggregation of liposomes when combined with acompound that is known not to activate chloride ion efflux in cellshaving the AF508 mutation, which compounds include 2-thio-CPX andisobutylmethylxanthine (IBMX), among others; (5) having a biphasicinteraction with adenosine wherein at lower adenosine concentrations of70 and 100 nM there was a modest increase in the aforementioned liposomeaggregation reaction driven by polypeptide Iα, at higher adenosineconcentrations such increase in activity was not seen, and at 1 μM andgreater the aggregation activity was suppressed; and (6) having anapparent KD of 69±27 nM with respect to CPX, a xanthine derivative knownto activate chloride ion efflux from ΔF508 cells.

[0049] The present invention also provides certain other CFTRpolypeptides that are contiguous to polypeptide Iα but, nevertheless,have characteristics that are significantly different. Polypeptide Iβ isa sequence of amino acids included in the CFTR protein from position 509to position 539, and has the following sequence: [SEQ ID NO:3]NH₂-GVSYDEYRYRSVIKACQLEEDISKFAEKDNI-COOH

[0050] Polypeptide Io is another sequence of amino acids included in theCFTR protein at from position 500 to position 530, and has the followingsequence: [SEQ ID NO:4] NH₂-GTIKENEEFGVSYDEYRYRSVIKACQLEEDIS-COOH

[0051] These polypeptides are contiguous or partially overlappolypeptide Iα, yet neither of these polypeptides drives the aggregationof chromaffin granules or synthetic liposomes, nor has any effect on theactivity of polypeptide Iα as disclosed herein or chloride ionefflux-activating xanthine derivatives. Analogously to the discussionabove relating to conservative amino acid changes of Iα, it iscontemplated that polypeptides having such conservative changes arefurther embodiments of the present invention.

[0052] The Iβ and Io polypeptides may be prepared and used as freepolypeptides, as fragments thereof, or associated with anothermacromolecule, as discussed hereinabove for the Iα polypeptide. The Iβand Io polypeptides may be used in affinity chromatography, and used asa negative control relative to the Iα polypeptide.

[0053] As with the Iα polypeptide discussed above, suitable sequences ofRNA and/or DNA corresponding to Iβ and Io may be identified by reversetranslation/transcription of the amino acid sequences of the respectivepolypeptide, using the genetic code. Accordingly, any suitable means maybe applied for generating the RNA molecules having any of the followingsequences: Iβ: [SEQ ID NO:5] GGN GUN UCN UAY GAY GAR UAY CGN UAY CGN UCNGUN AUH AAR GCN UGY CAR UUR GAR GAR GAY AUH UCN AAR UUY GCN GAR AAR GAYAAY AUH

[0054] Io: [SEQ ID NO:6] GGN ACN AUH AAR GAR AAY GAR GAR UUY GGN GUN UCNUAY GAY GAR UAY CGN UAY CGN UCN GUN AUH AAR GCN UGY CAR UUR GAR GAR GAYAUH UCN

[0055] Analogously to the Iα sequence, other sequences that encode thesame or functionally equivalent sequences as compared to SEQ ID NO:5 andSEQ ID NO:6 are contemplated as well. For example, the triplets UCN,GCN, and UUR may be replaced by AGY, AGR, and CUN, respectively, withoutany resultant amino acid sequence differences. In addition, nucleotidesequences that result in neutral amino acid charges, as discussed abovewith respect to the Iα sequences, are contemplated as additionalembodiments of the present invention. Moreover, as with the Iα sequence,the RNA complement of SEQ ID NO:5 and SEQ ID NO:6, as well as the DNAcounterpart sequences predicated thereon, are included as embodiments ofthe present invention, and may be determined by the application of theconventional genetic code by an ordinary artisan.

[0056] The aforementioned Iα, Iβ and Io polypeptides or their functionalequivalents may be used in a method for identifying a CFTR-bindingcompound. Such-a method comprises contacting a putative CFTR-bindingcompound to polypeptide Iα [SEQ ID NO:1] under conditions sufficient toallow for binding of the putative CFTR-binding compound to saidpolypeptide Iα, and determining, by any suitable means, whether suchbinding occurred, which would be indicative that the putativeCFTR-binding compound was indeed a CFTR-binding compound. ByCFTR-binding compound, it is intended that the compounds to beidentified by the present method will interact with a cell's CFTR,preferably a mutated CFTR such as ΔF508, such that the chloride ionefflux mediated by the CFTR will be increased, and perhaps restored.Such interaction between a CFTR-binding compound and the CFTR proteinmay be provided by any suitable combination of ionic, hydrophobic, andvan der Waals bonds or forces, as a function of the sequence of theamino acids of the CFTR and the physico-chemical characteristics of theCFTR-binding compound. For the identification of such CFTR-bindingcompounds, preferably those that are candidates for testing asCF-directed therapeutic agents, it is useful to test for positive effectof affinity to polypeptide Iα and, preferably, also for neutral ornegative effect of no affinity to polypeptide Iβ and/or Io. Theconditions used for such affinity assays for identifying CFTR-bindingcompounds can be optimized using conventional techniques well known inthe art. The method may identify any suitable compound that selectivelybinds to Iα, for example, but not to Iβ, for example, wherein thecompound is a hydrocarbon, including, but not limited to, a protein, alipid, a nucleic acid, or any combination thereof.

[0057] Among the effects of a CFTR-binding compound of interest are thefollowing: (1) commences or causes the aggregation of syntheticpolysaccharide liposomes when added to a preparation of (i.e.,composition comprising) such liposomes (described in Example 1); (2)commences or causes the aggregation of chromaffin granules when added toa preparation of (i.e., composition comprising) such chromaffin granules(described in Example 1); and (3) commences or causes the aggregation ofthe aforementioned liposomes at an increased rate when an activator ofchloride ion efflux of a ΔF508 cell is added to such liposomes (seeFIGS. 5-7). Activators of chloride ion efflux useful in the context ofthe present invention include CPX, caffeine,N,N-diallylcyclohexylxanthine, and other suitable compounds. No chlorideion efflux activator that has been tested in conjunction with Iα hasfailed to exhibit the phenomenon of increased rate of liposomeaggregation relative to the effect of Iα alone.

[0058] The present invention further provides a method of treating cellshaving a reduced apical Cl⁻ conductance, such as cystic fibrosis cells,and provides novel compounds and pharmaceutical compositions thereofuseful in the context of the present inventive method. Specifically, thetreatment method involves contacting cells having a reduced apical Cl⁻conductance with a therapeutically effective quantity of a compound thathas little or no affinity for adenosine cell receptors, particularlyeither the A₁-adenosine cell receptor or the A₂-adenosine cell receptor,yet stimulates Cl⁻ efflux. In particular, the method involves contactingsuch cells with a compound having the formula

[0059] wherein R₁ and R₃ are the same and are C₁-C₆ alkyl or C₁-C₆alkenyl, R₇ is C₁-C₆ alkyl or hydrogen, and R₈ is C₄-C₈ cycloalkyl, or atherapeutically effective derivative thereof. Such compounds may becharacterized with respect to their respective affinity for an adenosinereceptor, such as the A₁- or A₂-adenosine receptor. In particular, theaffinities of such compounds can be expressed with respect to a K_(i)value, such as having a K_(i) of greater than or equal to 0.01.Preferred compounds have a K_(i) of at least 0.05. Such preferredcompounds include those where R₁ and R₃ are methyl, propyl or allyl, R₇is methyl or hydrogen, and R₈ is cyclopentyl or cyclohexyl, and inparticular are one of the group consisting of1,3-dipropyl-7-methyl-8-cyclohexylxanthine,1,3-dipropyl-7-methyl-8-cyclopentylxanthine,1,3-diallyl-8-cyclohexylxanthine, and 8-cyclohexyl caffeine.

[0060] The present inventive method has particular utility in thetreatment of cystic fibrosis cells. The method is especially preferredin the treatment of cystic fibrosis cells that have a deletion involvingphenylalanine at amino acid position 508 of the cystic fibrosistransmembrane regulator, in particular those cystic fibrosis cells foundwithin a mammal, such as a human patient.

[0061] The compound used in the present inventive method is preferablyone that does not have phosphodiesterase activity. It is also preferredthat the therapeutically effective quantity of the compound is nontoxic.Most preferably, the compound, itself, is nontoxic or, if toxic,provides a benefit that outweighs such toxicity. It is further preferredthat the compound of the present invention has a low level of affinityfor any adenosine receptor (such as having a K_(i) of greater than orequal to about 0.01 mM) as recited above and exemplified below withrespect to A₁- or A₂-adenosine receptors. With respect to A₃-adenosinereceptors, it is not likely the compounds of the present invention havesubstantial affinity thereto because most xanthines cannot beaccommodated in the A₃-adenosine receptor binding site. van Galen etal., Medicinal Res. Rev., 12, 423-471 (1992). Especially preferredcompounds for use in the present inventive method are1,3-dipropyl-7-methylcyclopentylxanthine (DP-CPX),1,3-diallyl-8-cyclohexylxanthine (DCHX), and cyclohexyl caffeine (CHC);the most preferred compound is DCHX.

[0062] Such compounds and pharmaceutical compositions containing same,heretofore undisclosed, are themselves embodiments of the presentinvention. In particular, compounds of the present invention firstdisclosed herein have the formula

[0063] wherein (a) R₁ and R₃ are the same and are methyl or allyl, R₇ isethyl, cyclopropylmethyl or hydrogen, and R₈ is cyclohexyl, providedthat R₁ is allyl when R₇ is hydrogen and R₁ is methyl when R₇ is ethylor cyclopropylmethyl, or (b) R₁ and R₃ are both methyl, and R₇ ishydrogen or methyl, and R₈ is cyclohexylmethyl or cycloheptyl. Apreferred compound according to the aforementioned structure is compound17, i.e., 1,3-diallyl-8-cyclohexylxanthine (DCHX).

[0064] Alternatively, or additionally, a pharmaceutically acceptablederivative of DP-CPX, CHC, or DCHX, for example, or combinations thereofas yet further examples, may be used in the present inventive method,which provide yet another embodiment of the present invention. It isdesirable that such a derivative have equivalent therapeuticeffectiveness in the context of the present inventive method oftreatment.

[0065] The compound useful in the present inventive method may beadministered by any suitable means. One skilled in the art willappreciate that many suitable methods of administering the compound toan animal in the context of the present invention, in particular ahuman, are available, and, although more than one route may be used toadminister a particular compound, a particular route of administrationmay provide a more immediate and more effective reaction than anotherroute.

[0066] The compound is preferably administered directly to the lung of apatient. Preferably, the compound is administered as a pharmaceuticallyacceptable aqueous solution. It is even more preferable that thecompound be administered as an aqueous pharmaceutical solutioncontaining from about 0.001 to about 0.01%-w/w of the compound. Apharmaceutically acceptable aerosol is another preferred means ofadministration. The aerosol preferably contains from about 0.001 toabout 0.01% w/w of the compound.

[0067] The compound also may be administered orally. In such a case, thecompound will be generally administered in an amount of from about 0.1to 1 mg/kg body weight per day. A preferred amount of the inventivecompound for oral administration is about 0.1 mg/kg body weight per day.Other routes of administration, such as intravenous and intraperitonealadministration, are also possible.

[0068] The compound should be administered such that a therapeuticallyeffective concentration of the compound is in contact with the affectedcells of the body. The dose administered to an animal, particularly ahuman, in the context of the present invention should be sufficient toeffect a therapeutic response in the animal over a reasonable period oftime. The dose will be determined by the strength of the particularcompound employed and the condition of the animal, as well as the bodyweight of the animal to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particular compoundand the particular route of administration employed with a particularpatient. In general, the compounds of the present invention aretherapeutically effective at low doses. The effective dosage range isfrom about 30 nM to about 100 nM in the blood. Accordingly, thecompounds will be generally administered in relatively low doses.

[0069] The compound may be administered in a pharmaceutically acceptablecarrier. The present invention encompasses pharmaceutical compositionscomprising the present inventive compounds and pharmaceuticallyacceptable carriers. Pharmaceutically acceptable carriers are well-knownto those who are skilled in the art. The choice of carrier will bedetermined in part by the particular compound, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of the pharmaceuticalcomposition of the present invention.

[0070] Formulations suitable for oral administration include (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water or saline, (b) capsules, sachets or tablets,each containing a predetermined amount of the active ingredient, assolids or granules, (c) suspensions in an appropriate liquid, and (d)suitable emulsions. Tablet forms may include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels and the like containing, in addition to the activeingredient, such carriers as are known in the art.

[0071] Formulations suitable for administration by inhalation includeaerosol formulations placed into pressurized acceptable propellants,such as dichlorodifluoromethane, propane, nitrogen, and the like. Theactive agent may be aerosolized with suitable excipients.

[0072] Formulations suitable for intravenous and intraperitonealadministration, for example, include aqueous and nonaqueous, isotonicsterile injection solutions, which can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and nonaqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared for sterile powders,granules, and tablets of the kind previously described.

[0073] The desirable extent of the induction of Cl⁻ efflux from cellswill depend on the particular condition or disease being treated, aswell as the stability of the patient and possible side effects. Inproper doses and with suitable administration of certain compounds, thepresent invention provides for a wide range of activation of the rate ofchloride ion efflux, e.g., from little activation to essentially fullactivation.

[0074] The present invention is expected to be effective in thetreatment of all conditions, including diseases, that may becharacterized by a reduced cellular apical Cl⁻ conductance. Inparticular, the present invention is expected to have utility in thetreatment of chronic obstructive pulmonary diseases, especially cysticfibrosis.

[0075] The following examples serve to further illustrate the presentinvention and are not intended to limit the scope of the invention.

EXAMPLE 1

[0076] This example illustrates the methods used to assess themembrane-directed activity of certain CFTR polypeptides, and the resultsthereof.

[0077] Liposome aggregation assay: Phosphatidylserine (“PS”, obtainedfrom Avanti) was dissolved in chloroform, and small unilamellarliposomes were prepared by the extrusion process known in the art. ALipex machine was used to extrude the PS in a buffer consisting of 10 mMNa-HEPES, pH 7.2 and 1 mM EDTA. The liposomes were diluted to a finaloptical density at 540 nm of 0.01 and incubated with differentconcentrations of the different polypeptides and/or chloride ionefflux-activating compounds.

[0078] Chromaffin granule aggregation assay: Adrenal glands wereobtained from a local abattoir and transported to the laboratory on icewithin 2 hours of slaughter. Tissue was processed as described inBrocklehurst et al. in Polypeptide Hormones: A Practical Approach(Oxford Univ. Press, K. Siddle and J. Hutton, eds., 1990) at pp.237-255. Chromaffin granules were purified on a step gradient ofmetrizamide using standard procedures. After resuspension, the granuleaggregation assay was performed as described (Brocklehurst et al.,supra), and the synexin activity was assessed from the initial rate ofoptical density increase at 540 nm. Briefly, the assay was carried outat room temperature in a 1 ml quartz cuvette containing the chromaffingranule suspension in 0.3 mol/l sucrose, 40 mmol/l histidine buffer, pH6.0, synexin and variable amounts of calcium chloride.

[0079] Preparation of CFTR polypeptides: Polypeptides were synthesizedat the Yale University Department of Molecular Biology and Biophysics bystandard solid phase methods. The polypeptides were purified by reversephase HPLC, and the sequences were verified by direct chemicalsequencing, amino acid analysis, and by mass spectroscopy, usingstandard methods well-known in the art. The specific polypeptides wereIα [SEQ ID NO:1], Iβ [SEQ ID NO:3], and Io [SEQ ID NO:4].

[0080] Aggregation of chromaffin granules and liposomes by Iα [SEQ IDNO:1]: As shown in FIG. 2, polypeptide Iα was found to drive aggregationof bovine chromaffin granules in the presence of 1 mM CaCl₂. Bycontrast, the contiguous polypeptide Iβ [SEQ ID NO:3] was found to beessentially inactive in the chromaffin granule aggregation assay.Similarly, the overlapping polypeptide, Io [SEQ ID NO:4], was also foundto be inactive. These data thus appear to indicate that the action of Iαis specific to the Iα sequence.

[0081] A titration of the Iα activity, illustrated in FIG. 3A, showsthat the k_(1/2,app) was 22.2 μg/ml or 6.3 μM. A Hill plot of these data(see FIG. 3B) indicated that the Hill coefficient (n_(H)) was 2.6.Similar data were observed using phosphatidylserine liposomes as anaggregation target, and a detailed titration over the pH range betweenpH 5.5 and 7.5 indicated that the pH for 50% activity was ca. 6.75 (seeFIG. 4A).

[0082] Aggregation of PS liposomes by low concentrations of Iα: As shownin FIG. 2, the aggregation reactions at high polypeptide concentrationswere initiated promptly, and followed an apparent first order processthat effectively plateaued within 5-10 minutes. The cooperativecharacter of the aggregation reaction driven by Iα is quantitativelymanifest by the Hill coefficient (see FIG. 3A), which graphically showsthat at lower polypeptide concentrations the initial rates ofaggregation became small rather abruptly. However, at much longer timeperiods it became evident that a significant aggregation reaction stilloccurred, albeit with complex kinetics. As shown in FIG. 4B, thekinetics involved an initial lag phase of some 8-10 minutes, followed bya rising phase and a plateau. The conversion from an apparent firstorder process to the more complex process occurred below 2 μM of Iαpolypeptide, and the complex processes were clearly evident in a dosedependent manner between 0.4 and 2 μM. Below about 0.4 μM, all activityvirtually ceased.

[0083] Accordingly, Iα drives the above-described membrane aggregationphenomena whereas the adjacent (Iβ) or partially overlapping (Io)polypeptide sequence, with respect to CFTR, does not drive suchaggregation. The effective internal negative control clearly illustratesthe existence of a special functional property located on the CFTR atthe Iα site, which property clearly implicates membrane involvement.

EXAMPLE 2

[0084] This example illustrates the specific binding of certain chlorideion efflux activating compounds to Iα, and the inability of certaincompounds that do not activate chloride ion efflux to bind to Iα.

[0085] CPX was used as a representative compound known to activatechloride ion efflux from ΔF508 mutant cells. Stock solutions of CPX wereprepared in DMSO in the millimolar concentration range. The Iαpolypeptide (100 nM final polypeptide concentration) was dissolved in100 μl of a buffer solution containing 50 mM TRIS (pH=7.0), 300 mMsucrose, and 10 μM glutathione. After 1 hour of incubation at 37° C.,1-100 nM ³H-CPX (ICN, specific activity=109 C_(i)/mole) was added andincubated for a further 15 minutes. For displacement studies, unlabeledCPX, or other xanthine antagonists and nucleotides, were added to theincubation mixture before the radioactive probe. The mixture was thenfiltered through a nitrocellulose (NC) membrane (Schleicher & Schuell)by using a standard dot blot apparatus. Non-specific binding of ³[H]-CPXto NC filters was reduced by blocking filters with 0.3%polyethyleneimine (PEI). The dot blots were then washed five times with200 μl TRIS buffer-NaCl solutions (50 mM TRIS pH=7.0, 150 mM NaCl),radioactive spots were cut out, and the total was radioactivitydetermined by liquid scintillation counting. Specific ³H-CPX binding toIα was determined by subtracting the non-specific binding, defined bypreincubation with 10 AM unlabelled CPX, from total binding.

[0086] The binding of ³H-CPX to Iα was a saturable function of the CPXconcentration. A fit of the data to a Scatchard plot showed theK_(D,app) to be 69+27 nM (n=4). The B_(max), estimated from theScatchard plot intercept, indicated that the mole fraction of bound Iαpolypeptide was less than 1/2000. Displacement of binding was effectedby CPX, caffeine, and DAX, all known chloride ion efflux activators, butwas not effected by IBMX or 2-thio-CPX, which are known not to activatechloride ion efflux.

[0087] These data indicate that a chloride ion efflux activator can bindspecifically to Iα, thereby suggesting that the mechanism of CPXactivation of cells bearing the CFTR (ΔF508) mutation may be byinteraction with the CFTR molecule itself. Moroever, because of thespecificity of chloride ion efflux activators to bind and non-chlorideion efflux activators not to bind to Iα, these data illustrate theutility of this method to identify further CFTR-binding proteins thatmay have a therapeutic effect on CF cells.

EXAMPLE 3

[0088] This example illustrates the effect of CPX on PS liposomeaggregation driven by Iα.

[0089] The impact of CPX on PS liposome aggregation that was driven byIα was investigated. The study was done using concentrations of CPS ofbetween 1 and 30 nM (activation noted in ΔF508 containing cells),between 30 and 100 nM (maximum activation noted in the aforementionedcells), on up to 1000 nM (inhibition of activation noted in theaforementioned cells).

[0090] As shown in FIG. 5, liposome aggregation driven by 0.5 μg/ml(0.18 μM) Iα alone was very modest. However, with increases in CPXconcentration, the aggregation reaction was progressively activatedafter a lag of approximately 5 minutes. The maximum rate and extent ofaggregation was observed at 100 nM CPX, and it thereafter fell when theCPX concentration was raised to 300 μM. The inset to FIG. 5 shows theextent of the reaction at a 20 minute time point.

[0091] Accordingly, these data show a parallel between theconcentration-dependent behavior of CPX with (1) ΔF508 containing cellsand (2) Iα aggregation.

EXAMPLE 4

[0092] This example illustrates the effect of other xanthines on PSliposome aggregation driven by Iα. Included in the study were certainxanthine derivatives known to activate chloride ion efflux in ΔF508(e.g., caffeine and N,N-diallylcyclohexylxanthine (DAX)) or not toactivate such chloride ion efflux (e.g., 2-thio-CPX andisobutylmethylxanthine (IBMX)).

[0093] Results from a PS liposome aggregation assay are shown in FIG. 6.As is apparent from the graph, caffeine activates Iα aggregation withlow potency, and does not appear to inactivate Iα aggregation at thehighest concentrations. The inset to FIG. 6 shows the extent of thereaction at the 20 minute time point, such that the aggregation does notlevel off at all over the caffeine concentrations tested.

[0094] In the case of DAX, the potency is in the range of CPX, but noinactivation is seen at concentrations as high as 300 nM (see FIG. 7).The inset to FIG. 7 shows the extent of the reaction at a 20 minute timepoint. From this perspective, one can compare both CPX and DAX on thesame type of plot (FIG. 8), whereupon it can be seen that DAX is atleast one-half a log less potent than CPX. However, the efficacies withrespect to the chloride ion efflux of DAX and CPX are virtually thesame. Higher concentrations of DAX were not possible because ofsolubility limits.

[0095] Inclusion of 2-thio-CPX and IBMX, compounds known to be inactiveas activators of chloride ion efflux from cells bearing the ΔF508mutation, in the liposome aggregation assay showed these compounds to beentirely inactive at potentiating liposome aggregation driven by Iα atconcentrations up to 1 and 15 μM, respectively.

[0096] Accordingly, the Iα-driven PS liposome aggregation assay providesexactly parallel results with xanthine derivatives known to be active orinactive at chloride ion efflux activation.

EXAMPLE 5

[0097] This example illustrates the effect of adenosine on Iα-driven PSliposome aggregation.

[0098] Adenosine was included in a PS liposome aggregation assay.Adenosine had a modest but evident biphasic effect on the Iα-drivenliposome aggregation reaction. At the lower concentrations of 70 and 100nM, adenosine modestly activated the aggregation reaction. However, athigher concentrations, the activating effect was lost. At concentrationsof 1 μM adenosine and greater the aggregation reaction was actuallysuppressed below the already low control levels. These data thusindicate that the xanthines, CPX and DAX apparently interact with a siteon the Iα polypeptide with affinity for adenosine.

EXAMPLE 6

[0099] This example illustrates the synthesis and chemical analysis ofnovel xanthine derivatives of the present invention as well as of thosepreviously disclosed.

[0100] Alkylation of the N-7 position in xanthine derivatives to providecompounds 13, 17, 21, and 23 was carried out as described in Jacobson etal;, J. Med. Chem., 36, 2639-2644 (1993) and Daly et al., J. Med. Chem.,29, 1305-1308 (1986). Compounds 8-12, 14, 18, 19, 20, and 25 weresynthesized as described in Jacobson et al. (Biochem. Pharmacol., 37,3653-3661 (1988)); Shimada et al. (J. Med. Chem., 35, 924-930 (1992));and Shamim et al. (J. Med. Chem., 32, 1231-1237 (1989)); compounds 7,14, 18, and 19 have been disclosed in Mullet et al. (J. Med. Chem., 36,3341-3349 (1993)) and Shamin et al. (J. Med. Chem., 35, 924-930 (1989)).Compounds 1-5 and 26, as well as 2-chloroadenosine, were purchased fromResearch Biochemicals International (Natick, Mass.). Compound 27 wasdisclosed in Thompson et al. (J. Med. Chem., 34, 2877-2882 (1991)).TABLE 1 Conc. R₁, K_(i), K_(i), max. % thresh % Compound R₃ = R₇ = R₈ =μM(A₁) μM(A₂) (nM) control (nM) control 8-unsubstituted  1^(a) Me H H8.5 25 100 150 ± 30 1 130 ± 20  2 Me Me H 29 48 10 170 ± 20 1 120 ± 30 3^(b) Me, H H 7 16 >>10⁴ — — — iBu^(c) 8-cyclopentyl  4^(d) Me Hcyclopentyl 0.011 1.4 >>10⁴ — — —  5^(e) Pr H cyctopentyl 0.00046 0.3430 200 ± 10 1 130 ± 10  6 Pr Me cyclopentyl 2.3 11.4 ± 1.4 1000 180 ± 4030 125 ± 30  7 Pr, H cyclopentyl 0.014 0.58 30 160 ± 40 1 130 ± 20 H^(f) 8^(g) Pr H cyclopentyl 0.00066 0.31 >>10⁴ — — —  9 Pr H3-fluorocyclopentyl 0.042 — >>300 — — — 10 Pr H 3-iodocyclopentyl 0.058— >>300 — — — 11 Pr H cyclopenten-3-yl 0.045 0.640 ± 0.061 >>300 — — —12 Pr H noradamantyl 0.0013 0.38 10 170 ± 20 1 120 ± 5  8-cyclohexyl 13Me H cyclohexyl 0.030 ± 0.015 1.04 ± 0.20 3 160 ± 20   0.1 150 ± 30 14Me Me cyclohexyl 28 17.1 3 150 ± 30 3 150 ± 30 15 Me Et cyclohexyl 6.66± 0.99 3.23 ± 0.36 >>10⁴ — — — 16 Me cyclopropyl- cyclohexyl 8.23 ± 1.624.53 ± 1.37 >>10⁴ — — — methyl 17 allyl H cyclohexyl 0.656 ± 0.0189 4.94± 0.68 10³ 260 ± 70 30  170 ± 40 18 Pr H cyclohexyl 0.0015 0.423 ± 0.055>>10⁴ — — — 19 Pr Me cyclohexyl 2.7 9.24 ± 0.37 5 170 ± 50 5 170 ± 50 20Pr H cyclohexen-3-yl 0.010 0.905 ± 0.128 10⁴ — — — 21 Me Hcyclohexylmethyl 0.610 ± 0.076 5.47 ± 0.86 >>10⁴ — — — 22 Me Mecyclohexylmethyl 3.71 ± 0.36 5.46 ± 0.47 >>10⁴ — — — 8-cycloheptyl 23 MeH cycloheptyl 0.0659 ± 0.0145 1.05 ± 0.19 >>10⁴ — — — 24 Me Mecycloheptyl 19.5 ± 2.5  3.27 ± 0.98 >>10⁴ — — — 8-stryryl or 8-aryl25^(i) Me Me 3-chlorostyryl 28 0.054 30 160 ± 30 30 160 ± 30 26^(j) Pr Hp-φ-OCH₂CONH(CH₂)₂NH₂ 0.0012 0.08 >>10⁴ — — — non-xanthine antagonist 279-ethyl-6-cyclopenyladenine 0.44 17 >>10⁴ — — —

[0101] An alkyl or cycloalkyl carboxylic acid was reacted with5,6-diamino-1,3-dimethyluracil to obtain the corresponding amide,including 5-(cycloheptanoylamino)-6-amino-1,3-methyluracil and5-(cyclohexylmethylcarbonyl)-6-amino-1,3-methyluracil, using thefollowing protocol:

[0102] The acid (1 equiv) was dissolved in a minimum volume ofN,N-dimethylformamide (DMF) containing 1,3-dialkyl-5,6-diaminouracil(1.5 equiv). 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide:HCl (1equiv) was added, followed by a catalytic amount (0.05 equiv) of4-(N,N-dimethylamino)pyridine and 0.05 equiv of imidazole. The mixturewas stirred at room temperature for 3 hours, and saturated sodiumchloride solution was added (for 1,3-dipropyl derivatives, water wasused here) to form a precipitate or amorphous insoluble fraction. Theinsoluble residue was filtered and dissolved in 4 N aqueous sodiumhydroxide containing sufficient methanol to obtain a clear solution. Themixture was heated at 60° C. for 2 hours or until the completedisappearance of starting material, as judged using TLC (silica plate,CHCl₃; CH₃OH; HOAc; 85:10:5 v/v). The mixture was cooled and acidifiedto pH 1 with 6 N aqueous hydrochloric acid solution. The precipitate waswashed with water, dried, and further purified using a preparativesilica plate (85-95% CHCl₃; 5-15% methanol; 1-5% HOAc).

[0103] The new compounds were characterized (and resonances assigned) by300 MHz proton nuclear magnetic resonance mass spectrometry using aVarian GEMINI-300 FT-NMR spectrometer. Unless noted, chemical shifts areexpressed as ppm downfield from tetramethylsilane. New compounds werecharacterized by chemical ionization mass spectrometry (NH₃) on a massspectrometer or in the El mode using a VG7070F mass spectrometer.Representative spectral data for compounds 15, 16, 22, and 24 are: ¹HNMR CDCl₃ δ 3.42 (S, 3H N₃CH₃); 3.63 (S, 3H N₅CH₃); 3.89 (S, 6H OCH₃);5.06 (S, 2H, OCH₂); 6.8 (S, 2H); 6.78 (d, 1H, J=16 Hz); 7.3-7.5 (m, 5H);7.7 (D, 1H, J=16 Hz); MS (Cl) m/e 463 (MH⁺ base), 375, 357.

[0104] Carbon, hydrogen, and nitrogen analyses were carried out byAtlantic Microlabs (Norcross, Ga.), and ±0.4% was acceptable. A summaryof the characterization studies conducted pursuant to application of theaforementioned techniques is provided in Table 2, where the formulas andelemental analyses of compounds 15-17 and 21-24 are presented. TABLE 2Calculated Found Compound Yield % M.p.(C) Formula C H N C H N 15 81 >170C₁₅H₂₂N₄O₂.1/4H₂O 61.10 7.69 19.00 61.25 7.48 18.59 16 91 >120C₁₇H₂₄N₄O₂.1/4H₂O 63.63 7.70 17.46 63.62 7.57 17.16 17 — 126-128C₁₇H₂N₄O₂.1/2H₂O 63.38 7.08 17.28 63.14 7.17 17.32 21 74 236-238C₁₄H₂₀N₄O₂ 60.85 7.30 20.27 60.77 7.32 20.22 22 73 158-159C₁₅H₂N₄O₂.0.15H₂O 61.48 7.67 19.12 61.76 7.83 18.72 23 84 223-225C₁₄H₂₀N₄O₂ 60.85 7.30 20.27 60.67 7.31 20.22 24 89 199-201 C₁₅H₂N₄O₂62.05 7.64 19.30 61.97 7.68 19.24

[0105] All xanthine derivatives were judged to be homogeneous using thinlayer chromatography prior to biological testing.

EXAMPLE 7

[0106] This example describes the measurement of chloride efflux fromCFPAC cells in response to contact between such cells and the xanthinederivatives of the present invention, including protocols required forculturing such cells and implementing such experimentation thereon.

[0107] CFPAC cells are pancreatic adenocarcinoma cells that arehomozygous for the most common CFTR mutation, i.e., deletion of Phe⁵⁰⁸(Schoumacher et al., Proc. Natl. Acad. Sci., 87, 4012-4016 (1990)).CFPAC cells and CFTR-transfected CFPAC cells (CFPAC-4.7 CFTR) wereobtained from R. Frizzell at the University of Alabama. The cells weresplit and seeded at low density on 24-well COSTAR plates in mediumcomposed of Iscove's medium, supplemented with 10% (vol/vol)heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 mg/mlstreptomycin, 0.25 mg/ml fungizone, and 1% (wt/vol) glutamine, andadjusted to an osmolarity of 310 mosm. All culture materials wereobtained from Biofluids (Rockville, Md.). After 5 hours, the medium wasreplaced, and attached cells were allowed to grow to confluency during aperiod of 48-72 hours at 37° C. in an atmosphere of 5% CO₂/95% air.

[0108] Before each experiment, cells were loaded with [³⁶Cl⁻] asfollows. Confluent cells were washed four times in the medium disclosedin Example 6. Then, after aspirating the last wash, 500 μl of mediumcontaining about 1.4×10⁸ counts per minute (cpm) of (36Cl)⁻ (Amersham)were added to each well. The plates of cells were then incubated at 37°C. overnight in 5% CO₂/95% air, thus allowing [³⁶Cl]⁻ isotopicequilibrium to be obtained. To initiate efflux experiments,concentrations of CPX and/or other xanthine derivatives were added tothe cells, which were then incubated for 15 minutes at 19° C. Afterincubating, the cells were washed four times in successive aliquots ofan ice-cold wash medium composed of 150 mM sodium-gluconate, 1.5 mMpotassium gluconate, and 10 mM Na-Hepes (pH 7.4). At the end of the washstep, 500 μl of bicarbonate-free flux medium at 19° C. was added, andsampling was initiated by collecting 50 μl aliquots from each well at 0,1, 2, 3, 5, 7, and 10 minutes. The flux medium consisted of 150 mMsodium gluconate, 1.5 mM potassium gluconate, 10 mM sodium Na-Hepes (pH7.4), 100 AM bumetamide to inhibit efflux due to the cotransporter, anddifferent concentrations of CPX or other drugs, e.g., A₁-adenosineantagonists and activators of cAMP synthesis, as required and describedin subsequent examples. The osmolarity was 310 mOsm. At the end of eachflux experiment, 20 μl of 50% trichloroacetic acid was added to a finalconcentration of 5% to obtain a measure of remaining radioactivity.Samples were mixed with 1.5 ml of Cytoscint fluid and assayed for twominutes on a Beckman LS9000 scintillation counter with windows atmaximum width. osmotic strength was measured by freezing pointdepression on an osmotic osmometer. See Eidelman et al., supra, 1992.

[0109] Table 1 summarizes the data derived from at least four repeats ofthe chloride efflux experiments using the xanthine derivatives of thepresent invention. Additionally, each data point within a repeatedexperiment was computed from the average of efflux measurementsperformed on four separate cells. The column labeled “Conc. max (nM)”presents the concentration of the xanthine derivative at its maximumimpact upon chloride efflux, and the succeeding column to the rightpresents the percentage of chloride efflux at maximum impact relative tocontrol. The column labeled “thresh (nM)” presents the minimumconcentration of the xanthine derivative required to detect an effectupon chloride efflux, and the succeeding column to the right presentsthe percentage of chloride efflux at minimum detection relative tocontrol. As can be seen in Table 4, the range of maximal stimulation isfrom about 3 nM (for compound 14, for example) to over 10⁴ nM (forcompound 3, for example). Percentage increases over control at themaximum stimulation concentrations ranged from about 10% (i.e.,non-effective compounds such as compounds 2 and 12) to about 260%+/−70%(for compound 17). The concentration of the xanthine derivative atthreshold stimulation of the chloride efflux ranged from 0.1 nM (forcompound 13, for example) to about 10⁴ nM (for compound 20, forexample). Percentage increases over control at the threshold stimulationconcentrations ranged from 120% (for compound 12) to about 170% (forcompound 17, for example).

[0110] From these data, and in view of the preference of using a lesserconcentration of any given drug to effect a desired result so as tominimize the incidence of undesired side effects, it is evident thatcompounds 6, 7, 13, 14, 17, 19, and 25 are preferred compounds in viewof their noted ability to stimulate chloride efflux at μM or lesserconcentrations.

[0111] FIGS. 9-12 present flux study data that is presented graphicallyfor compound 1, i.e., theophylline (FIG. 9); compound 5, i.e., CPX (FIG.10); compound 22, i.e., 8-cyclohexylmethyl caffeine (FIG. 11); andcompound 17, i.e., 1,3-diallyl-8-cyclohexylxanthine (FIG. 12). Thevertical axis in each of the graphs of FIGS. 9-12 represents the rate of[³⁶Cl]⁻ efflux (FA) of the radiolabeled chloride as a function of theconcentration of the compound tested (horizontal axis). These werecalculated by linear regression of the log (FA) as a function of time,using standard techniques, and are presented as a percentage relative tothe rate constants of control wells on the same plate. Data were handledwith a Quatro-Pro 4.0 program. The error bars represent the S.E.M.values of four experiments (i.e., n=4).

[0112] As can be seen by simple observation of FIGS. 9-12, each ofcompounds 1, 5, and 22 has the characteristic of inducing 200% of theefflux rate as compared to control. Compound 17 also demonstrates inexcess of 100% of the efflux capability of the control, but to a lesserextent as compared to the other three compounds. Of the four compoundswhose results are presented herein graphically, compounds 1 and 22 havelittle affinity for either tested adenosine receptor, compound 17 hasmoderate affinity for A₁ receptors (K_(i)≅0.07), and compound 5 hasextreme affinity for A₁ receptors (K_(i)≅0.0005). Despite the fact thatcompound 17 has significantly less affinity for A₁-receptors thancompound 5, for example, compound 17 has been shown to be significantlymore efficacious in its absolute chloride efflux capability, as noted inTable 1 (a maximum of 260% of control for compound 17 versus 200% ofcontrol for compound 5).

EXAMPLE 8

[0113] This example illustrates assays for the detection of adenosinereceptor binding and the results of such assays performed in partialcharacterization of the novel xanthine derivatives of the presentinvention.

[0114] Rat cerebral cortical membranes and striatal membranes wereprepared according to the procedure of Hide et al., Mol. Pharmacol., 41,352-359 (1992) and treated with adenosine deaminase (2 U/ml) for 30minutes at 37° C. prior to storage at −70° C. Solid samples of theadenosine derivatives were dissolved in dimethyl sulfoxide (DMSO) andstored in the art at. −20° C. The stock solutions were diluted with DMSOto a concentration of ≦0.1 mM prior to addition to the aqueous medium.The final concentration of DMSO in the assay medium was generally 2%.

[0115] Inhibition of binding of 1 nM [³H]N⁶-phenylisopropyladenosine(Dupont NEN, Boston, Mass.) to A₁ receptors in rat cerebral cortexmembranes was measured as described in Schwabe et al.,Naunyn-Schmiedeberg's Arch. Pharmacol., 313, 179-187 (1980), andJacobson et al., J. Med. Chem., 36, 1333-1342 (1993a). Membranes(^(˜)100 μg protein per tube) were incubated for 1.5 hours at 37° C. ina total volume of 0.5 ml of 50 mM tris hydrochloride at pH 7.4. Testdrugs were dissolved in DMSO and added in 10 μl aliquots, resulting in afinal DMSO concentration of 2%. Bound and free radioligand wereseparated by addition of 3 ml of a buffer containing 50 mM trishydrochloride, at pH 7.4 at 5° C., followed by vacuum filtration using aBrandel Cell Harvester (Brandel, Gaithersburg, Md.) and a Whatman GF/Bglass fiber filter with additional washes totaling 9 ml of buffer.Non-specific binding was determined with 10 μM 2-chloroadenosine.

[0116] Inhibition of binding of 5 nM [³H]CGS 21680(2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine)(Dupont NEN, Boston, Mass.) to A2a receptors was carried out as reportedin Jarvis et al., J. Pharmacol. Exp. Therap., 251, 888-893 (1989).Membranes (^(˜)80 μg protein per tube) were incubated for one hour at25° C. in a total volume of 0.5 ml of a buffer solution consisting of 50nM tris hydrochloride and 10 mM MgCl₂ at pH 7.4. Test drugs weredissolved in DMSO and added in 10 μl aliquots, resulting in a final DMSOconcentration of 2%. Non-specific binding was defined using 20 μM2-chloroadenosine. Filtration was carried out using a Brandel CellHarvester, as above, using the tris.HCl/MgCl₂ buffer recited hereinaboveas the washing buffer.

[0117] At least six different concentrations spanning three orders ofmagnitude, adjusted appropriately for the IC₅₀ of each compound, wereused. IC₅₀ values, computer-generated using a non-linear regressionformula on the (GraphPAD program (Institute for Scientific Information),were converted to apparent K_(i) values using K_(D) values (Jacobson etal., supra, 1993a; Ukena et al., FEBS Letters, 209, 122-128 (1986)) of1.0 and 14 nM for [³H]PIA and [³H]CGS 21680 binding, respectively, andthe method of Cheng et al. (Biochem. Pharmacol., 22, 3099-3108 (1973)).

[0118] The results of the adenosine receptor binding studies arepresented in Table 1 for all of the xanthine derivatives under thecolumns “K_(i), μM (A₁)” and “K_(i), μM (A₂).” Receptor antagonistshaving a high affinity for a particular receptor are characterized byhaving very low K_(i) values, as determined using the assays describedabove for the A₁ and A₂ receptors. Accordingly, it can be seen thatcompound 5 has high affinity for the Al receptor whereas compound 6 haslow affinity for either of the receptors tested.

[0119] FIGS. 13-14 also depict data derived from the receptor studiesdescribed above, with particular reference to ascertaining the effectsof changing ring size of the 8-cycloalkyl substituent on binding atadenosine receptors. Values for rat cortical Al-receptors (closedcircles, ) and rat striatal A_(2a)-receptors (open triangles, Δ) areincorporated into the graphs. The affinity of 8-cycloalkyltheophyllinederivatives (FIG. 16) and the affinity of 8-cycloalkylcaffeinederivatives (FIG. 17) at A₁- and A₂-receptors are shown. Values aregiven as the average of two or three (+/−s.e.m.) determinationsperformed in triplicate. Data for 8-cyclobutyl derivatives are fromJacobson et al., J. Med. Chem., 36, 2639-2644 (1993b).

[0120]FIGS. 13 and 14 demonstrate that among derivatives of theophylline(1,3-dimethylxanthine), the cyclopentyl analogue, compound 4, had thehighest A₁ affinity. Among derivatives of caffeine(1,3,7-trimethylxanthine), the cycloheptyl analogue, compound 24, showedsome A_(2a) affinity (6-fold), whereas smaller ring sizes (4-6) hadlittle to no affinity (Jacobson et al., supra, 1993b).

[0121] These structure activity relationships diverge greatly from thoseobserved for the same compounds in stimulating Cl⁻ efflux.

[0122] The results provided above, when considered in toto, indicatethat compounds 6, 14, 17, and 19 provide the characteristics ofsubstantial efflux activity at low concentration ranges coupled to alack of activity at the adenosine receptors.

[0123] The results of these examples indicate that it is possible to (1)identify compounds that activate cellular chloride ion efflux using anin vitro liposome- and/or CFTR-binding assay; and (2) activate chlorideefflux from CF cells by using a compound that has little or no affinityfor the A₁- or the A₂-adenosine receptor, such as compounds 6, 14, 17,or 19. The most efficacious compound tested was compound 17. Thetherapeutic advantages of a drug that is able to activate chlorideefflux from CF cells specifically without having an impact orsubstantial impact at adenosine receptors are appreciable, given thatsuch drug action would be less encumbered by undesirable side effects toother tissues.

[0124] All of the references identified herein, including patents,patent applications, and publications, are hereby incorporated byreference in their entireties.

[0125] While the invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations in the preferred method, compound, andcomposition can be used and that it is intended that the invention canbe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications encompassedwithin the spirit and scope of the invention as defined by the followingclaims.

1 6 32 amino acids amino acid single linear polypeptide 1 Pro Ser GluGly Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser 1 5 10 15 Gln PheSer Trp Ile Met Pro Gly Thr Ile Lys Glu Asn Glu Glu Phe 20 25 30 96 basepairs nucleic acid single linear mRNA 2 CCNUCNGARG GNAARAUHAA RCAYUCNGGNCGNAUHUCNU UYUGYUCNCA RUUYUCNUGG 60 AUHAUGCCNG GNACNAUHAA RGARAAYGARGARUUY 96 31 amino acids amino acid single linear polypeptide 3 Gly ValSer Tyr Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys 1 5 10 15 GlnLeu Glu Glu Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile 20 25 30 32amino acids amino acid single linear polypeptide 4 Gly Thr Ile Lys GluAsn Glu Glu Phe Gly Val Ser Tyr Asp Glu Tyr 1 5 10 15 Arg Tyr Arg SerVal Ile Lys Ala Cys Gln Leu Glu Glu Asp Ile Ser 20 25 30 93 base pairsnucleic acid single linear mRNA 5 GGNGUNUCNU AYGAYGARUA YCGNUAYCGNUCNGUNAUHA ARGCNUGYCA RUURGARGAR 60 GAYAUHUCNA ARUUYGCNGA RAARGAYAAY AUH93 96 base pairs nucleic acid single linear mRNA 6 GGNACNAUHA ARGARAAYGARGARUUYGGN GUNUCNUAYG AYGARUAYCG NUAYCGNUCN 60 GUNAUHAARG CNUGYCARUURGARGARGAY AUHUCN 96

What is claimed is:
 1. A polypeptide selected from the group comprisingpolypeptide Iα [SEQ ID NO:1], polypeptide Iβ [SEQ ID NO:3], andpolypeptide Io [SEQ ID NO:4].
 2. The polypeptide of claim 1, whereinsaid polypeptide is linked covalently to an affinity chromatographymatrix.
 3. The polypeptide of claim 2, wherein said affinitychromatography matrix is sepharose, agarose, polyacrylamide, orcellulose.
 4. A method for identifying a CFTR-binding compoundcomprising contacting a putative CFTR-binding compound with polypeptideIα [SEQ ID NO:1] under conditions sufficient to allow for binding ofsaid putative CFTR-binding compound to said polypeptide Iα, anddetermining whether such binding occurred, an indication that saidputative CFTR-binding compound is a CFTR-binding compound.
 5. The methodof claim 4, wherein said CFTR-binding compound has a neutral ornegative-affinity for polypeptide Iβ [SEQ ID NO:3] or polypeptide Io[SEQ ID NO:4].
 6. The method of claim 4, wherein said CFTR-bindingcompound causes the aggregation of liposomes when added to a compositioncomprising said liposomes.
 7. The method of claim 4, wherein saidCFTR-binding compound causes the aggregation of chromaffin granules whenadded to a composition comprising said chromaffin granules.
 8. Themethod of claim 7, wherein said composition consists essentially of saidchromaffin granules in 1 mM CaCl₂.
 9. A method of treating cysticfibrosis in a mammal in need of such treatment, comprising administeringa therapeutically effective amount of a compound having the formula

wherein R₁ and R₃ are the same and are C₁-C₆ alkyl or C₁-C₆ alkenyl, R₇is C₁-C₆ alkyl or hydrogen, and R₈ is C₄-C₈ cycloalkyl, and wherein theK_(i) with respect to said compound and an adenosine receptor is atleast 0.01.
 10. The method of claim 9, wherein R₁ and R₃ are methyl,propyl, or allyl, R₇ is methyl or hydrogen, and R₈ is cyclopentyl orcyclohexyl.
 11. The method of claim 9, wherein said mammal is a human.12. The method of claim 11, wherein said compound is selected from thegroup consisting of 1,3-dipropyl-7-methyl-8-cyclopentylxanthine,1,3-dipropyl-7-methyl-8-cyclohexylxanthine,1,3-diallyl-8-cyclohexylxanthine, and 8-cyclohexyl caffeine.
 13. Themethod of claim 12, wherein said compound is1,3-diallyl-8-cyclohexylxanthine.
 14. The method of claim 9, whereinsaid compound is administered to the lung of said human.
 15. The methodof claim 14, wherein said compound is administered as an aqueouspharmaceutical composition containing from about 0.001 to about 0.01%w/w of said compound.
 16. The method of claim 9, wherein said K_(i) isat least 0.05.
 17. A compound having the formula

wherein (a) R₁ and R₃ are the same and are methyl or allyl, R₇ is ethyl,cyclopropylmethyl or hydrogen, and R₈ is cyclohexyl, provided that R₁ isallyl when R₇ is hydrogen and R₁ is methyl when R₇ is ethyl orcyclopropylmethyl, or (b) R₁ and R₃ are both methyl, and R₇ is hydrogenor methyl, and R₈ is cyclohexyl-methyl or cycloheptyl.
 18. The compoundof claim 17, wherein said compound is 1,3-diallyl-8-cyclohexylxanthine.19. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a compound having the formula

wherein (a) R₁ and R₃ are the same and are methyl or allyl, R₇ is ethyl,cyclopropylmethyl or hydrogen, and R₈ is cyclohexyl, provided that R₁ isallyl when R₇ is hydrogen and R₁ is methyl when R₇ is ethyl orcyclopropylmethyl, or (b) R₁ and R₃ are both methyl, and R₇ is hydrogenor methyl, and R₈ is cyclohexylmethyl or cycloheptyl.
 20. Thepharmaceutical composition of claim 19, wherein said compound is1,3-diallyl-8-cyclohexylxanthine.
 21. A polynucleotide selected from thegroup consisting of polynucleotide Iα [SEQ ID NO:2], polynucleotide Iβ[SEQ ID NO:5], and polynucleotide Io [SEQ ID NO:6].